Frequency domain photon migration (FDPM) was used to non-invasively quantify optical properties (absorption and scattering) of cervical tissues during photodynamic therapy (PDT). The aims of this study were 1) to quantitatively determine near-infrared optical properties of normal cervical tissues and high-grade squamous intraepithelial lesions (HSIL), 2) to assess the feasibility of differentiating normal cervical tissues from HSIL on the basis of these optical properties, and 3) to determine how cervical tissue optical properties change following PDT of HSIL in vivo. Eleven patients were scheduled for photodynamic therapy of histologically proven high-grade cervical squamous intraepithelial lesion with ALA (5-aminolevulinic acid). FDPM measurements were performed on normal cervical tissues and HSIL prior to ALA application onto the ecto-cervix, ninety minutes after ALA administration, and three minutes after completion of PDT light delivery. FDPM measurements of no rmal cerv ical tissues were compared to HSIL using two-tailed analysis of variance (ANOVA). The results of this study clearly demonstrate that FDPM is 1) sensitive to PDT induced changes in cervical optical properties (absorption changes up to 20%), 2) absorption and scattering are significantly reduced in dysplastic cervical tissue vs. normal (reduction in scattering ranges from 6-13% and absorption from 8-16%), and 3) dysplastic processes induce wavelength dependent alterations in absorption and scattering. These results suggest that near-infrared FDPM may have an important role in non-invasive detection of cervical dysplasia.